Atrazine (ATR, 2-chloro-4-(ethylamino)-6-(isopropyl-amino)-1,3,5-triazine) has been one of the most widely applied herbicides in the US and Midwestern states. An estimated 36.3 million kg of ATR is applied annually to more than 69% of all U.S. corn acreage (United States Department of Agriculture, 2004). The contamination of surface and ground water by ATR and its chlorinated metabolites has raised public health and ecological concerns recently due to their endocrine disruption activities and potential risk of gastroschisis (Hayes et al., 2006; Waller et al., 2010). The ATR and its metabolites are persistent in the environmental and the mineralization of ATR and its chlorinated metabolites or complete cleavage of the triazine ring in the environment is limited to less than 2-10%. A 2006 study by the U.S. Geological Survey found ATR and its metabolites were detected in approximately 75 percent of stream water and about 40 percent of all groundwater samples from agricultural areas tested between 1992 and 2001 (Gilliom et al., 2006).
Despite the persistence of ATR and its metabolites in the VBS environment (Lin et al., 2005), the enzymes including chlorohydrolase AtzA produced by naturally occurring degradative bacteria Pseudomonas sp. strain ADP rapidly hydrolyzes the atrazine to the much less toxic and less mobile metabolite hydroxyatrazine. The catabolic genes that encode for the enzymes responsible for each step in the ATR degradation process have been well characterized (FIG. 1). Pseudomonas sp. strain ADP can utilize ATR and its metabolites as a carbon source and sole nitrogen source (Mandelbaum et al., 1995; Martinez et al., 2001). This property is due to the presence of the pADP-1 plasmid, a 108-kDA catabolic plasmid which encodes for all the metabolic enzymes necessary to completely degrade ATR to CO2 and NH3 (De Souza et al., 1998a; Martinez et al., 2001). The AtzA-C enzymes are not unique to Pseudomonas sp. strain ADP, but are found among soil bacteria isolates across the U.S. and Europe (De Souza et al., 1998b). The atzA chlorohydrolase metalloenzyme not only has the ability to dechlorinate atrazine into the significantly less toxic hydroxyatrazine, but also shows degradative activity to other s-triazines, such as simazine and desethylatrazine (Boundy-Mills et al., 1997; De Souza et al., 1996). Catabolic gene atzB metabolizes hydroxyatrazine to N-isopropylammelide, whose hydrolytic deamidation to cyanuric acid and isopropylamine is catalyzed by atzC (Boundy-Mills et al., 1997; Sadowsky et al., 1998). Gene atzD encodes a cyanuric acid amidohydrolase, which converts cyanuric acid to biuret. The presence of the atzDEF operon is unique to Pseudomonas sp. strain ADP and allows this bacterium to further catabolize the cyanuric acid (produced by the activities of AtzA-C) into CO2 and NH3 (Martinez et al., 2001). Biuret hydrolase is encoded by atzE and allophanate hydrolase is encoded by atzF, resulting in the conversion of biuret to allophanate and allophanate to CO2 and NH3 (Martinez et al., 2001). This plasmid is highly transmittable between microorganisms, and the expression of these genes, particularly atzD and F, are sensitive to alternative N sources in the environment (atz+ to atz−) (Garcia-Gonzalez et al., 2003). The purification and optimization of ATR enzymes for large scale remediation of organic pesticides including atrazine were first commercialized by CSIRO Enzyme Based Bioremediation Technology. However, the persistence of the enzymes such as AtzN has short life under the field conditions. The enzymatic activities cannot be sustained more than 8-10 days.
The recent success of enzyme conjugation technology and ordered mesoporous material synthesis (Hartmann, 2005) has allowed the encapsulation of enzymes and other biomolecules into ordered mesoporous material. According to IUPAC definition, material containing pores with diameters ranging from 2 to 50 nm are classified as ordered mesoporous material. The modified ordered mesoporous carbon material have high specific surface area, large specific pore volume, regular arrays of uniform nanopores, and narrow pore size of distribution which provide a large adsorption capacities and unique temples for functional modification. To immobilize the bioactive biomolecules or enzymes on the surface of the ordered mesoporous material, the surface of porous materials usually needs to be modified and functionalized. The functionalization of the materials usually carries out by reacting with organic or inorganic oxidative agents, such as nitric acid, ozone, or ammonium persulfate, followed by the substitution of oxidative groups by functionalities containing amine (—NH2), thiol (R—SH) or free sulfhydryl (—SH) (Contescu, 1998; Hartmann, 2005; Jarrais, 2005; Puri, 1971; Tamai, 2006a). The functionalization processes provide the functional groups required for conjugation of biomolecules or enzymes. Hypothetically, the ordered mesoporous materials immobilized with bioactive enzymes or biomolecules should not only retain their functional enzymatic or biological characteristics by the enzymes/biomolecules immobilized on the surface, but also exhibit high specific surface areas and large adsorption capacities for the organic pollutants, like atrazine, through electrostatic interactions and/or covalent bindings.
Therefore, there is a need to provide a new and improved series of enzymatic delivery compounds with improved surface specificity and adsorption capacity and the ability to retain the enzymes' biological characteristics during delivery.